Method of amplifying nucleic acid sequences

ABSTRACT

The invention is directed to methods of removing amplicons of non target and/or target nucleic acid sequences having one or more modified (e.g., methylated) nucleotides from a sample wherein the sample comprises the non target nucleic acid and a target nucleic acid sequence to be amplified.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/341,540, filed Jul. 25, 2014, which is a continuation ofInternational Application No. PCT/US2013/063931, which designated theUnited States and was filed on Oct. 8, 2013, published in English andclaims the benefit of U.S. Provisional Application No. 61/729,072, filedon Nov. 21, 2012. The entire teachings of the above applications areincorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL IN ASCII TEXT FILE

This application incorporates by reference the Sequence Listingcontained in the following ASCII text file being submitted concurrentlyherewith:

-   -   a) File name: 47231011005SEQUENCELISTING.txt; created Apr. 14,        2016, 3 KB in size.

BACKGROUND OF THE INVENTION

Many clinical labs rely on uracil DNA glycosylase (UDG) (also known asuracil N-glycosylase (UNG)) decontamination of polymerase chain reaction(PCR) products. Amplicons containing uracil as opposed to thymidine canbe digested with UDG to eliminate any residual PCR product in thelaboratory. Many Next Generation sequencing platforms utilizepolymerases that cannot traverse a uracil (utilize polymerases that areuracil illiterate). For instance, the Illumina MiSeq and HiSeq platformsrely on polymerases with proof reading activity to generate the seededclusters for surface PCR. Proof reading polymerases such as pfu willstall on uracils in the template strand. Due to this, amplification ofuracilyated templates fail to initiate cluster PCR thus eliminating thepotential of UDG decontamination methods.

Thus, improved amplification methods and/or decontamination ofamplification methods are needed for nucleic acid amplificationtechniques such as PCR.

SUMMARY OF THE INVENTION

In some aspects, the invention is directed to a method of removingamplicons of non target nucleic acid sequence having one or moremodified (e.g., methylated) nucleotides from a sample wherein the samplecomprises the non target nucleic acid sequence and a target nucleic acidsequence to be amplified. The method comprises contacting the samplewith a composition comprising a restriction enzyme that cleaves (e.g.,specifically (selectively) cleaves (recognizes)) a nucleic acid sequencecomprising the modified nucleotides (e.g., a methyl specific restrictionenzyme) and that is capable of being deactivated, thereby producing acombination; maintaining the combination under conditions in which theamplicons of the non target nucleic acid are digested by the restrictionenzyme (e.g., methyl specific restriction enzyme) prior to amplificationof the target nucleic acid; and amplifying the target nucleic acidsequence thereby producing amplicons of the target nucleic acidsequence, and thereby removing the amplicons of the non target nucleicacid from the sample comprising the target nucleic acid sequence to beamplified.

In other aspects, the invention is directed to a method of seriallyamplifying a target nucleic acid sequence wherein the firstamplification is performed with a first cleavable base and a subsequentamplification is performed with a second cleavable base, and the firstcleavable base and the second cleavable base are different. In someaspects, the first cleavable base is cleaved by a first restrictionenzyme and the second cleavable base is a uniquely cleavable base thatis cleaved by a second restriction enzyme that specifically cleavesamplicons comprising the uniquely cleavable base; the subsequentamplification is performed with the uniquely cleavable base and thesecond restriction enzyme; and the first amplification is performed withthe first cleavable base wherein amplicons comprising the firstcleavable base can be simultaneously cleaved with the first restrictionenzyme that cleaves the different cleavable base.

In other aspects, the invention is directed to a method of removingamplicons of non target nucleic acid sequence having one or morenucleotides that are modified (e.g., methylated) from a sample whereinthe sample comprises the non target nucleic acid sequence and a targetnucleic acid sequence to be serially amplified. The method comprisescontacting the sample with a composition (a first composition)comprising (i) deoxynucleotide triphophates (dNTPs) comprising dATP,dTTP, dGTP, and dCTP wherein one or more of the deoxynucleotidetriphophates are modified (e.g., modified with a first moiety, e.g.,methylated with a first methyl group) (ii) a nucleic acid polymerase,(iii) one or more primers that are complementary to a portion of thetarget nucleic acid sequence, and (iv) a first restriction enzyme (e.g.,a (first) methyl specific restriction enzyme) that is capable of beingdeactivated and that digests nucleic acid sequences comprisingnucleotides modified with the first moiety (e.g., that are methylatedwith the first methyl group), thereby producing a combination (a firstcombination). The combination is maintained under conditions in whichthe amplicons of the non target nucleic acid are digested by the firstrestriction enzyme (e.g., a methyl specific restriction enzyme) prior toamplification of the target nucleic acid. The target nucleic acidsequence is amplified, thereby producing amplicons of the target nucleicacid sequence having one or more modified nucleotides comprising thefirst moiety (e.g., nucleotides that are methylated with the firstmethyl group). The amplicons of the target nucleic acid sequence arecontacted with a composition (second composition) comprising (i)deoxynucleotide triphophates (dNTPs) comprising dATP, dTTP, dGTP, anddCTP wherein one or more of the deoxynucleotide triphophates aremodified (e.g., modified with a second moiety, e.g., methylated with asecond methyl group), (ii) a nucleic acid polymerase, (iii) one or moreprimers that are complementary to a portion of the target nucleic acidsequence, and (iv) a second restriction enzyme (e.g., a (second) methylspecific restriction enzyme) that is capable of being deactivated andthat selectively digests nucleic acid sequences comprising nucleotidesmodified with the second moiety (e.g., that are methylated with thesecond methyl group), thereby producing a combination (a secondcombination). The combination is maintained under conditions in whichthe amplicons of the non target nucleic acid are digested by the secondrestriction enzyme (e.g., the second methyl specific restriction enzyme)prior to amplification of the target nucleic acid. The target nucleicacid sequence is amplified, thereby producing amplicons of the targetnucleic acid sequence having one or more nucleotides modified with thefirst moiety and the second moiety (amplicons of the target nucleic acidsequence that are methylated with the first methyl group and the secondmethyl group), thereby removing amplicons of the non target nucleic acidhaving one or more nucleotides that are modified (e.g., methylated) froma sample wherein the sample comprises the non target nucleic acid and atarget nucleic acid sequence to be serially amplified.

In other aspects, the invention is directed to a method of removingamplicons of a target nucleic acid sequence after amplification of thetarget nucleic acid sequence. The method comprises contacting the targetnucleic acid sequence with (i) deoxynucleotide triphophates (dNTPs)comprising dATP, dTTP, dGTP, and dCTP wherein one or more of thedeoxynucleotide triphophates are modified (e.g., methylated) (ii) anucleic acid polymerase, and (iii) one or more primers that arecomplementary to a portion of the target nucleic acid sequence, therebyproducing a combination. The combination is maintained under conditionsin which the target nucleic acid is amplified, thereby generatingamplicons of the target nucleic acid sequence wherein one or more of theamplicons comprise one or more of the modified (e.g., methylated)nucleotides. The amplicons are contacted with a restriction enzyme(e.g., a methyl specific restriction enzyme) that digests nucleic acidsequences comprising the modified nucleotides, thereby removing the oneor more amplicons which comprise one or more of the modified (e.g.,methylated) nucleotides.

In other aspects, the invention is directed to a method of amplifying atarget nucleic acid sequence. The method comprises contacting the targetnucleic acid sequence with native nucleotides, a nucleic acidpolymerase, and one or more primers wherein each primer is complementaryto a portion of the target nucleic acid sequence and comprises one ormore modified (e.g., methylated) nucleotides, thereby producing acombination. The combination is maintained under conditions in which thetarget nucleic acid is amplified, thereby generating amplicons of thetarget nucleic acid sequence wherein one or more of the ampliconscomprise one or more modified (e.g., methylated) nucleotides. Theamplicons are contacted with a restriction enzyme (e.g., a methylspecific restriction enzyme), thereby removing all or a portion of theprimers from the one or more amplicons which comprise one or more of themodified (e.g., methylated) nucleotides.

In other aspects, the invention is directed to a method of replicating asingle stranded oligo or DNA library. The method comprises ligating afirst amplification primer to the single stranded oligo library or DNAlibrary thereby forming a ligation product. The ligation product iscontacted with a modified (e.g., methylated) primer that hybridizes tothe first amplification primer and a polymerase, thereby forming acombination. The combination is maintained under conditions in which areverse complement of the single stranded oligo or DNA is generated. Asecond amplification primer is ligated to the reverse complement,thereby producing a double adapted ligation product sequence. The doubleadapted litigation product is contacted with native nucleotides, anucleic acid polymerase, and one or more primers wherein each primer iscomplementary to a portion of the double adapted litigation productsequence and comprises one or more modified (e.g., methylated)nucleotides, thereby producing a combination. The combination ismaintained under conditions in which the double adapted litigationproduct is amplified, thereby generating amplicons of the double adaptedlitigation product sequence wherein one or more of the ampliconscomprise one or more of the modified (e.g., methylated) nucleotides. Theamplicons are contacted with a restriction enzyme (e.g., a methylspecific restriction enzyme) that cleaves nucleotide sequencescomprising one or more of the modified nucleotides, thereby removing allor a portion of the primers from the one or more amplicons whichcomprise one or more modified (e.g., methylated) nucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: A biotinylated probe library often used in exome capture ortargeted sequencing is represented. 3′ OH is targeted with a singlestranded DNA Ligase like Circligase I or Circligase II (Epicentre) orMth Ligase (NEB). A Universal Primer with a 3′ cap is ligated to theprobe library. The cap is required to prevent Primer to Primer ligation.

FIG. 2: 2nd Strand synthesis is performed with the complement to the M13universal primer with a methylated CNNR signal for subsequent MspJIdigestion. Polymerase extension double strands the probe library andleaves only one 3′ OH which can targetted with the 2nd Ligation step.Once this final ligation step is complete, PCR with Methylated primerscan be performed. This shares some similarities with 5 prime independentcloning described by Pak and Fire but has the added benefit of beingable to subsequently remove the amplification primers to bring the oligolibrary back to its native state after amplification.

FIG. 3: Representation of an oligo Library after amplification. It has 2methylated CNNR signals in the PCR primers. It has an optional Biotinthat can help to single strand the oligo library after amplification. Ithas no internal methylation signals (green arrow) in this embodiment butPCR with methylCTP is an option for decontamination procedures. The bluearrows represent the cut sites after amplification and digestion byMspJI.

FIG. 4: Depiction of the MspJI restriction activity (SEQ ID NOs: 1 and2) as described by Zheng et al. Nucleic Acids Res 38(16): 5527-5534.

FIGS. 5A-5C: 5A shows results of PCR with uracil replacement; 5B sowsresults of PCR control with native dNTPs; 5C shows results of digestionwith UDG, FpG (sample 1=no UTP, sample 2=dUTP library.

FIG. 6: Methylated amplification

FIG. 7: Digested libraries

FIG. 8: Graph pf PhiX library amplified with dCTP and 5′ me dCTP.

FIG. 9: Electropherogram of amplification with methyl dCTP

FIG. 10: Electropherogram of amplification with native dCTP

FIG. 11: Clustering results

FIG. 12: Results of EpiSEEK patients sequences ith and without 5-methyldCTP.

FIG. 13: Déjà vu PCR makes use of what is termed herein a “DNA diode”where enzymes that specifically digest 5th and 6th bases respectivelyare leveraged to ensure complex serial amplification steps can beperformed contamination free without physical isolation of labequipment. Red dots are Hydroxyl groups, Green dots are Hydrogen, Bluedots are Carbon, thus hydroxymethyl groups have 1 red, 2 green, 1 bluedot while methyl cytosine, have 3 Green dots and 1 Blue dot.

FIG. 14: Observed vs Expected coverage of a mitochondrial DNA deletedsample mixed with a known full length (16.6 kb) wild type mtDNA sample.4.5 kb Kearns-Sayre homozygous mtDNA deletion was then diluted into awildtype 16.6 Kb barcoded mitochondrial sample at known mixture ratios,barcoded and sequenced on an ILMN Miseq V2 sequencer. Expected coverageof the known undeleted region vs the observed ratio of these regions wasascertained by barcode demultiplexing and read counting. This result wasexpected in that a multiplexed 12 Kb PCR proceeds at a more rapid ratethan its 16.6 kb PCR competitive product despite 15 minute extensiontimes applied in PCR. This also highlighted the pronounced sensitivityfor detecting large deletions in mtDNA samples using LR-PCR.

FIG. 15: Secondary PCR of Nextera libraries using 5me-dCTP (Green,Turqoise) and 5hme-dCTP (Red, Blue). 16.6 kb amplicons were fragmentedwith Nextera at 55° C. for 30 minutes. Subsequent PCR utilized 12 cyclesof amplification using Q5 polymerase (NEB) with additional nucleotidesspiked in.

FIG. 16: Following amplification, 10 ul of product (estimated 80 ng) wasdigested with 5 Units of AbaSI for 1.5 hrs at 25° C. with a 65° C. 20min heat kill. As suggested by Wang et al, cleavage with AbaSI appearsspecific to 5hydroxymethylcytosine fragments.

FIG. 17: Use of DMSO is estimated to lower the Tm 0.6° C. per %according to Von Ashen et al. This improves the C20 coverage of targetsin sequencing panels.

FIG. 18: Ratio of Mitochondrial reads to Nuclear reads using Methyldigestion with MspJI and Methyl enrichment with Methyl Binding Domains.

FIG. 19: To measure decontamination potential we spiked in 5me-dCTPamplified mtDNA from a different haplogroup into Target mtDNA to beamplified and measured heteroplasmy levels with and without MspJIdecontamination. MspJI digestion removed 100% of expected heteroplasmycontaminants suggesting it can decontaminate equimolar contaminationevents or less. Note: red bars are at 0% demonstrating completedecontamination at equimolar contamination levels.

FIG. 20: Haloplex 320 amplicon capture of Mitochondrial DNA providesvariable coverage. Long Range PCR makes has more uniform coverage andmore obvious deletion detection.

FIG. 21: SYBR Green Real Time PCR estimates mitochondrial copy number at416 copies next to diploid genes BECN1 and NEB.

DETAILED DESCRIPTION OF THE INVENTION

A description of example embodiments of the invention follows.

Universal primers are utilized in many clinical PCR applications. A sideeffect of universal primers is that subsequent PCR reaction setups areeasily contaminated with PCR products from a previous amplificationreaction. Clinical laboratories have traditionally utilized dUTP in PCRto generate PCR products that are different from genomic DNA and arespecifically cleavable with uracil DNA glycosylase (UDG).

To address the issues associated with decontamination methods fornucleic acid (e.g., DNA; RNA), the use of a single cycle of primerextension with adenosine (A), cytosine (C), guanine (G) and thymine (T)instead or uracil (U) will generate hemi-uracilyated amplicons where theWatson strand is void of uracils while the Crick strand remainsuracilyated. The first step in seeding the DNA cluster PCR requiresdenaturation (e.g., NaOH denaturization) where the Watson strand canoperate independently of the Crick strand and be utilized successfullyin cluster PCR. This delivers a clinical sequencing pipeline that iscongruent with UDG decontamination methods. Using these methods, onlythe PCR products that contain uracil are enzymatically digestedtherefore any contaminating PCR products can be digested with no risk todigesting the target DNA about to be amplified. Unfortunately,uracilated DNA is not amplified well with widely-used emulsion orcluster PCR kits, due to the use of uracil-illiterate polymerases inmost next generation sequencing platforms.

To address the issues associated with amplification methods usinguracilated DNA, in one aspect, the invention provides for use of one ormore modified (e.g., methylated) deoxynucleotide triphosphates (dNTPs)(e.g., deoxycytidine triphosphate (dCTP) such as methyl dCTP (5-methyldCTP; and 5-hydroxymethyl cystine) or methylated primers (e.g., a primercomprising methylated nucleotides) in conjunction with methyl specificrestriction enzymes, e.g., MspJ1 (New England Biolabs) (Zheng et al) toamplify a target nucleic acid sequence and/or remove amplicons of atarget nucleic acid sequence after amplification of the target nucleicacid sequence. The benefits of using methylated dNTPs is that it isincorporated more readily and with less error than dUTP and severalantibodies and methyl binding proteins are commercially available toisolate methylated PCR products from non-methylated PCR products.Examples and methods are described within demonstrating the success ofeach of these techniques.

In some aspects, described herein is a decontamination ready encodedamplification, referred to herein as “DREAM PCR”, that replaces thisuracil base with methylcytosine, as most polymerases are methylcytosineliterate and efficiently incorporate this base into a PCR product (REF).In other aspects, in addition to 5-methylcytosine, the recentlydescribed “6th base” 5-hydroxymethylcytosine and the enzymes that existwhich differentially digest or capture hydroxymethylated cytosine areused. As described herein, techniques that detect modified bases such as5-hydroxymethylcytosine from 5-methylcytosine, and that moreover,differentially detect 5-hydroxymethylcytosine from 5-methylcytosineprovide for improved amplification methods.

To enable selective serial digestion of the two nucleotides, DREAM PCRsubstitutes the methyl-specific endonuclease MspJI in place of UDG.MspJI digests heavily methylated PCR products differentially thanlightly methylated substrate genomic DNA and it has a preference fordigesting double stranded methylated DNA over single stranded lightlymethylated circular gDNA presented with a Haloplex exome capture system(McKernan in press). Incorporation of 5-hydroxymethylcytosine enablesserial PCR steps to be performed each with a different 5th base and eachrespectively digestable with unique enzymes (MspJI and AbaSI). Thisoffers unique decontamination solutions for more complex massivelyparallel DNA sequencing workflows requiring more than one amplificationstep.

Accordingly, in some aspects, the invention is directed to a method ofremoving amplicons of non target nucleic acid having one or moremodified (e.g., methylated) nucleotides from a sample wherein the samplecomprises the non target nucleic acid and a target nucleic acid sequenceto be amplified. The method comprises contacting the sample with acomposition comprising a restriction enzyme that cleaves (e.g.,specifically (selectively) cleaves (recognizes)) a nucleic acid sequencecomprising the modified nucleotide(s) (e.g., a methyl specificrestriction enzyme) and that is capable of being deactivated, therebyproducing a combination; maintaining the combination under conditions inwhich the amplicons of the non target nucleic acid are digested by therestriction enzyme (e.g., methyl specific restriction enzyme) prior toamplification of the target nucleic acid; and amplifying the targetnucleic acid sequence thereby producing amplicons of the target nucleicacid sequence, and thereby removing the amplicons of the non targetnucleic acid from the sample comprising the target nucleic acid sequenceto be amplified. The composition can further comprise (i)deoxynucleotide triphophates (dNTPs) comprising dATP, dTTP, dGTP, anddCTP (ii) a nucleic acid polymerase, (iii) one or more primers that iscomplementary to a portion of the target nucleic acid sequence.

In a particular aspect, the invention is directed to a method ofremoving amplicons of non target nucleic acid having one or moremethylated nucleotides from a sample wherein the sample comprises thenon target nucleic acid and a target nucleic acid sequence to beamplified. The method comprises contacting the sample with a compositioncomprising a methyl specific restriction enzyme that is capable of beingdeactivated, thereby producing a combination; maintaining thecombination under conditions in which the amplicons of the non targetnucleic acid are digested by the methyl specific restriction enzymeprior to amplification of the target nucleic acid; and amplifying thetarget nucleic acid sequence thereby producing amplicons of the targetnucleic acid sequence, and thereby removing the amplicons of the nontarget nucleic acid from the sample comprising the target nucleic acidsequence to be amplified. The composition can further comprise (i)deoxynucleotide triphophates (dNTPs) comprising dATP, dTTP, dGTP, anddCTP (ii) a nucleic acid polymerase, (iii) one or more primers that iscomplementary to a portion of the target nucleic acid sequence. Themethod can further comprise contacting the amplicons of the targetnucleic acid sequence with a (second, active) methyl specificrestriction enzyme, thereby producing a combination (a secondcombination) and maintaining the combination under conditions in whichthe amplkcons of the target nucleic acid sequence are digested. Themethyl specific restriction enzyme that is contacted with the ampliconsof the target nucleic acid sequence can be identical to the methylspecific restriction enzyme that is contacted with the amplicons of thenon target nucleic acid sequence or can be a different methyl specificrestriction enzyme than the methyl specific restriction enzyme that iscontacted with the amplicons of the non target nucleic acid sequence.The method can further comprise contacting the amplicons of the targetnucleic acid sequence with a methyl specific restriction enzyme prior toamplification of a second target nucleic acid.

In other aspects, the invention is directed to a method of seriallyamplifying a target nucleic acid sequence wherein the firstamplification is performed with a first cleavable base and a subsequent(e.g., second, third fourth, fifth, etc.) amplification is performedwith a second cleavable base, and the first cleavable base and thesecond cleavable base are different. In some aspects, the firstcleavable base is cleaved by a first restriction enzyme and the secondcleavable base is a uniquely cleavable base that is cleaved by a secondrestriction enzyme that specifically cleaves amplicons comprising theuniquely cleavable base; the subsequent amplification is performed withthe uniquely cleavable base and the second restriction enzyme; and thefirst amplification is performed with the first cleavable base whereinamplicons comprising the first cleavable base can be simultaneouslycleaved with the first restriction enzyme that cleaves the differentcleavable base. The method can further comprise contacting the targetnucleic acid sequence with (i) deoxynucleotide triphophates (dNTPs)comprising dATP, dTTP, dGTP, and dCTP (ii) a nucleic acid polymerase,(iii) one or more primers that is complementary to a portion of thetarget nucleic acid sequence.

In another aspect, the invention is directed to a method of seriallyamplifying a target nucleic acid sequence wherein the firstamplification is performed with a first cleavable base and a subsequent(e.g., a second, third, fourth, fifth, etc.) amplification is performedwith a second cleavable base, and the first cleavable base and thesecond cleavable base are different. That is, the cleavable bases differin that when present in a nucleic acid sequence (e.g., an amplicon) afirst cleavable base is cleaved by one (e.g., a first) restrictionenzyme and the second cleavable base is cleaved by another (e.g.,second, distinct) restriction enzyme. The method can further comprisecontacting the target nucleic acid sequence with (i) deoxynucleotidetriphophates (dNTPs) comprising dATP, dTTP, dGTP, and dCTP (ii) anucleic acid polymerase, (iii) one or more primers that is complementaryto a portion of the target nucleic acid sequence.

In addition, the method can comprise contacting the target nucleic acidwith a composition comprising (i) deoxynucleotide triphophates (dNTPs)comprising dATP, dTTP, dGTP, and dCTP wherein one or more of thedeoxynucleotide triphophates comprise the first cleavable base (ii) anucleic acid polymerase, (iii) one or more primers that arecomplementary to a portion of the target nucleic acid sequence, and (iv)the first restriction enzyme wherein the first restriction enzyme iscapable of being deactivated, thereby producing a combination; andmaintaining the combination under conditions in which nucleic acidsequences comprising the first cleavable base are digested by the firstrestriction enzyme prior to amplification of the target nucleic acid.The method can further comprise amplifying the target nucleic acidsequence under conditions in which amplicons of the target nucleic acidsequence have one or more of the first cleavable base; contacting theamplicons of the target nucleic acid sequence with a compositioncomprising (i) deoxynucleotide triphophates (dNTPs) comprising dATP,dTTP, dGTP, and dCTP wherein one or more of the deoxynucleotidetriphophates comprise the second cleavable base (ii) a nucleic acidpolymerase, (iii) one or more primers that are complementary to aportion of the target nucleic acid sequence, and (iv) the secondrestriction enzyme that is capable of being deactivated and thatselectively digests nucleic acid sequences comprising the secondcleavable base; maintaining the combination of b) under conditions inwhich nucleic acid sequences comprising the second cleavable base aredigested by the second restriction enzyme prior to amplification of thetarget nucleic acid; and amplifying the target nucleic acid sequence,thereby producing amplicons of the target nucleic acid sequencecomprising the first cleavable base and the second cleaveable base. Thefirst cleavable base can be methylated dCTP and the first restrictionenzyme is MspJI.

In other aspects, the invention is directed to a method of removingamplicons of non target nucleic acid having one or more nucleotides thatare modified (e.g., methylated) from a sample wherein the samplecomprises the non target nucleic acid and a target nucleic acid sequenceto be serially amplified. The method comprises contacting the samplewith a composition (a first composition) comprising (i) deoxynucleotidetriphophates (dNTPs) comprising dATP, dTTP, dGTP, and dCTP wherein oneor more of the deoxynucleotide triphophates are modified (e.g., modifiedwith a first moiety, e.g., methylated with a first methyl group) (ii) anucleic acid polymerase, (iii) one or more primers that arecomplementary to a portion of the target nucleic acid sequence, and (iv)a first restriction enzyme (e.g., a (first) methyl specific restrictionenzyme) that is capable of being deactivated and that digests nucleicacid sequences comprising nucleotides modified with the first moiety(e.g., that are methylated with the first methyl group), therebyproducing a combination (a first combination). The combination ismaintained under conditions in which the amplicons of the non targetnucleic acid are digested by the first restriction enzyme (e.g., amethyl specific restriction enzyme) prior to amplification of the targetnucleic acid. The target nucleic acid sequence is amplified, therebyproducing amplicons of the target nucleic acid sequence having one ormore modified nucleotides comprising the first moiety (e.g., nucleotidesthat are methylated with the first methyl group). The amplicons of thetarget nucleic acid sequence are then subsequently (serially) amplifiedby contacting the amplicons with a composition (second composition)comprising (i) deoxynucleotide triphophates (dNTPs) comprising dATP,dTTP, dGTP, and dCTP wherein one or more of the deoxynucleotidetriphophates are modified (e.g., modified with a second moiety, e.g.,methylated with a second methyl group), (ii) a nucleic acid polymerase,(iii) one or more primers that are complementary to a portion of thetarget nucleic acid sequence, and (iv) a second restriction enzyme(e.g., a (second) methyl specific restriction enzyme) that is capable ofbeing deactivated and that selectively digests nucleic acid sequencescomprising nucleotides modified with the second moiety (e.g., that aremethylated with the second methyl group), thereby producing acombination (a second combination). The combination is maintained underconditions in which the amplicons of the non target nucleic acid aredigested by the second restriction enzyme (e.g., the second methylspecific restriction enzyme) prior to the subsequent amplification ofthe target nucleic acid. The target nucleic acid sequence is amplified,thereby producing amplicons of the target nucleic acid sequence havingone or more nucleotides modified with the first moiety and the secondmoiety (amplicons of the target nucleic acid sequence that aremethylated with the first methyl group and the second methyl group),thereby removing amplicons of the non target nucleic acid having one ormore nucleotides that are modified (e.g., methylated) from a samplewherein the sample comprises the non target nucleic acid and a targetnucleic acid sequence to be serially amplified.

In other aspects, the invention is directed to a method of removingamplicons of non target nucleic acid having one or more nucleotides thatare methylated from a sample wherein the sample comprises the non targetnucleic acid and a target nucleic acid sequence to be seriallyamplified. The method comprises contacting the sample with a composition(a first composition) comprising (i) deoxynucleotide triphophates(dNTPs) comprising dATP, dTTP, dGTP, and dCTP wherein one or more of thedeoxynucleotide triphophates are methylated with a first methyl group(ii) a nucleic acid polymerase, (iii) one or more primers that arecomplementary to a portion of the target nucleic acid sequence, and (iv)a first methyl specific restriction enzyme that is capable of beingdeactivated and that digests nucleic acid sequences comprisingnucleotides that are methylated with the first methyl group, therebyproducing a combination (a first combination). The combination ismaintained under conditions in which the amplicons of the non targetnucleic acid are digested by the first methyl specific restrictionenzyme prior to amplification of the target nucleic acid. The targetnucleic acid sequence is amplified, thereby producing amplicons of thetarget nucleic acid sequence having one or more nucleotides that aremethylated with the first methyl group. In a subsequent amplification ofthe target nucleic acid sequence, the amplicons of the target nucleicacid sequence are contacted with a composition (second composition)comprising (i) deoxynucleotide triphophates (dNTPs) comprising dATP,dTTP, dGTP, and dCTP wherein one or more of the deoxynucleotidetriphophates are methylated with a second methyl group (ii) a nucleicacid polymerase, (iii) one or more primers that are complementary to aportion of the target nucleic acid sequence, and (iv) a second methylspecific restriction enzyme that is capable of being deactivated andthat selectively digests nucleic acid sequences comprising nucleotidesthat are methylated with the second methyl group, thereby producing acombination (a second combination). The combination is maintained underconditions in which the amplicons of the non target nucleic acid aredigested by the second methyl specific restriction enzyme prior toamplification of the target nucleic acid. The target nucleic acidsequence is amplified, thereby producing amplicons of the target nucleicacid sequence having one or more nucleotides that are methylated withthe first methyl group and the second methyl group, thereby removingamplicons of the non target nucleic acid having one or more nucleotidesthat are methylated from a sample wherein the sample comprises the nontarget nucleic acid and a target nucleic acid sequence to be seriallyamplified.

In some aspects of the method the dNTP methylated with the first methylgroup is methylated dCTP and the first methyl specific restrictionenzyme is MspJI. In other aspects of the method, the dNTP methylatedwith the second methyl group is hydroxymethylated dCTP and the secondmethyl specific restriction enzyme is AbaSI.

In other aspects, the invention is directed to a method of removingamplicons of a target nucleic acid sequence after amplification of thetarget nucleic acid sequence. The method comprises contacting the targetnucleic acid sequence with (i) deoxynucleotide triphophates (dNTPs)comprising dATP, dTTP, dGTP, and dCTP wherein one or more of thedeoxynucleotide triphophates are modified (e.g., methylated) (ii) anucleic acid polymerase, and (iii) one or more primers that arecomplementary to a portion of the target nucleic acid sequence, therebyproducing a combination. The combination is maintained under conditionsin which the target nucleic acid is amplified, thereby generatingamplicons of the target nucleic acid sequence wherein one or more of theamplicons comprise one or more of the modified (e.g., methylated)nucleotides. The amplicons are contacted with a restriction enzyme(e.g., a methyl specific restriction enzyme) that digests nucleic acidsequences comprising the modified nucleotides, thereby removing the oneor more amplicons which comprise one or more of the modified (e.g.,methylated) nucleotides.

In other aspects, the invention is directed to a method of removingamplicons of a target nucleic acid sequence after amplification of thetarget nucleic acid sequence. The method comprises contacting the targetnucleic acid sequence with (i) deoxynucleotide triphophates (dNTPs)comprising dATP, dTTP, dGTP, and dCTP wherein one or more of thedeoxynucleotide triphophates are methylated (ii) a nucleic acidpolymerase, and (iii) one or more primers that are complementary to aportion of the target nucleic acid sequence, thereby producing acombination. The combination is maintained under conditions in which thetarget nucleic acid is amplified, thereby generating amplicons of thetarget nucleic acid sequence wherein one or more of the ampliconscomprise one or more methylated nucleotides. The amplicons are contactedwith a methyl specific restriction enzyme, thereby removing the one ormore amplicons which comprise one or more methylated nucleotides.

In other aspects, the invention is directed to a method of amplifying atarget nucleic acid sequence. The method comprises contacting the targetnucleic acid sequence with native nucleotides (e.g., dATP, dTTP, dCTP,dGTP), a nucleic acid polymerase, and one or more primers wherein eachprimer is complementary to a portion of the target nucleic acid sequenceand comprises one or more modified (e.g., methylated) nucleotides,thereby producing a combination. The combination is maintained underconditions in which the target nucleic acid is amplified, therebygenerating amplicons of the target nucleic acid sequence wherein one ormore of the amplicons comprise one or more modified (e.g., methylated)nucleotides. In a particular aspect, the primers of the amplicon only orprimarily comprise one or more of the modified nucleotides. Theamplicons are contacted with a restriction enzyme (e.g., a methylspecific restriction enzyme), thereby removing all or a portion of theprimers from the one or more amplicons which comprise one or more of themodified (e.g., methylated) nucleotides.

In other aspects, the invention is directed to a method of amplifying atarget nucleic acid sequence. The method comprises contacting the targetnucleic acid sequence with native nucleotides, a nucleic acidpolymerase, and one or more primers wherein each primer is complementaryto a portion of the target nucleic acid sequence and comprises one ormore methylated nucleotides, thereby producing a combination. Thecombination is maintained under conditions in which the target nucleicacid is amplified, thereby generating amplicons of the target nucleicacid sequence wherein one or more of the amplicons comprise one or moremethylated nucleotides. In a particular aspect, the primers of theamplicon only or primarily comprise one or more of the methylatednucleotides. The amplicons are contacted with a methyl specificrestriction enzyme, thereby removing all or a portion of the primersfrom the one or more amplicons which comprise one or more methylatednucleotides. In some aspects, each primer comprises one or moremethylated cytosines, one or more methylated adenosines or a combinationthereof.

In other aspects, the invention is directed to a method of replicating asingle stranded oligo or DNA library. The method comprises ligating afirst amplification primer to the single stranded oligo library or DNAlibrary thereby forming a ligation product. The ligation product iscontacted with a modified (e.g., methylated) primer that hybridizes tothe first amplification primer and a polymerase, thereby forming acombination. The combination is maintained under conditions in which areverse complement of the single stranded oligo or DNA is generated. Asecond amplification primer is ligated to the reverse complement,thereby producing a double adapted ligation product sequence. The doubleadapted litigation product is contacted with native nucleotides, anucleic acid polymerase, and one or more primers wherein each primer iscomplementary to a portion of the double adapted litigation productsequence and comprises one or more modified (e.g., methylated)nucleotides, thereby producing a combination. The combination ismaintained under conditions in which the double adapted litigationproduct is amplified, thereby generating amplicons of the double adaptedlitigation product sequence wherein one or more of the ampliconscomprise one or more of the modified (e.g., methylated) nucleotides. Theamplicons are contacted with a restriction enzyme (e.g., a methylspecific restriction enzyme) that cleaves nucleotide sequencescomprising one or more of the modified nucleotides, thereby removing allor a portion of the primers from the one or more amplicons whichcomprise one or more modified (e.g., methylated) nucleotides.

In other aspects, the invention is directed to a method of replicating asingle stranded oligo or DNA library. The method comprises ligating afirst amplification primer to the single stranded oligo library or DNAlibrary thereby forming a ligation product. The ligation product iscontacted with a methylated primer that hybridizes to the firstamplification primer and a polymerase, thereby forming a combination.The combination is maintained under conditions in which a reversecomplement of the single stranded oligo or DNA is generated. A secondamplification primer is ligated to the reverse complement, therebyproducing a double adapted ligation product sequence. The double adaptedlitigation product is contacted with native nucleotides, a nucleic acidpolymerase, and one or more primers wherein each primer is complementaryto a portion of the double adapted litigation product sequence andcomprises one or more methylated nucleotides, thereby producing acombination. The combination is maintained under conditions in which thedouble adapted litigation product is amplified, thereby generatingamplicons of the double adapted litigation product sequence wherein oneor more of the amplicons comprise one or more methylated nucleotides.The amplicons are contacted with a methyl specific restriction enzyme,thereby removing all or a portion of the primers from the one or moreamplicons which comprise one or more methylated nucleotides. In someaspects, a template independent DNA ligase is used to ligate methylatedamplification primers to the oligo library. In other aspects, thetemplate independent DNA ligase is Mth Ligase. In yet other aspects,each primer comprises one or more methylated cytosines, one or moremethylated adenosines or a combination thereof.

As used herein, “amplifying” “amplification” or an “amplificationreaction” refers to methods for amplification of a nucleic acid sequenceincluding polymerase chain reaction (PCR), ligase chain reaction (LCR),rolling circle amplification (RCA), strand displacement amplification(SDA) and multiple displacement amplification (MDA), serialamplification as will be understood by a person of skill in the art.Such methods for amplification comprise, e.g., primers that anneal tothe nucleic acid sequence to be amplified, a DNA polymerase, andnucleotides. Furthermore, amplification methods, such as PCR, can besolid-phase amplification, polony amplification, colony amplification,emulsion PCR, bead RCA, surface RCA, surface SDA, etc., as will berecognized by one of skill in the art. It will also be recognized thatit is advantageous to use an amplification method that results inexponential amplification of free DNA molecules in solution or tetheredto a suitable matrix by only one end of the DNA molecule. Methods thatrely on bridge PCR, where both PCR primers are attached to a surface(see, e.g., WO/18957 and Adessi et al., Nucleic Acids Research (2000):28(20): E87) result in only linear amplification, which does not producesufficient amounts of product to support efficient library constructionfor subsequent sequencing. Furthermore, the products of bridge PCRtechnologies are array-bound, and would have to be cleaved from thesupport as intact double stranded DNA molecules to be useful forsubsequent sequencing. In addition, it will be recognized that it isoften advantageous to use amplification protocols that maximize thefidelity of the amplified products to be used as templates in DNAsequencing procedures. Such protocols use, for example, DNA polymeraseswith strong discrimination against misincorporation of incorrectnucleotides and/or strong 3′ exonuclease activities (also referred to asproofreading or editing activities) to remove misincorporatednucleotides during polymerization.

The methods provided herein utilize a (one or more) modified bases. Asused herein, the term “base” refers to the heterocyclic nitrogenous baseof a nucleotide or nucleotide analog (e.g., a purine, a pyrimidine, a7-deazapurine). A “nucleoside” refers to a nitrogenous base linked to asugar molecule. A “nucleotide” (e.g., “deoxyribonuleotide (dNTP)”,“ribonucleotide”) is a nitrogenous heterocyclic base (or nucleobase),which can be either a double-ringed purine or a single-ringedpyrimidine; a five-carbon pentose sugar (deoxyribose in DNA or ribose inRNA); and a phosphate group. Suitable bases for use in the methods ofthe invention include, but are not limited to, adenine (A) (e.g., dATP),cytosine (C) (e.g., dCTP), guanine (G) (e.g., dGTP), thymine (T) (e.g.,dTTP), and uracil (U) (e.g., dUTP). These and other suitable bases willpermit a nucleotide bearing the base to be enzymatically incorporatedinto a polynucleotide chain. The base will also be capable of forming abase pair involving hydrogen bonding with a base on another nucleotideor nucleotide analog. The base pair can be either a conventional(standard) Watson-Crick base pair or a non-conventional (non-standard)non-Watson-Crick base pair, for example, a Hoogstein base pair orbidentate base pair. The terms “base” and “deoxynucleotide triphiosphate(dNTP)” are at times used interchangeably.

As used herein, “Watson-Crick base pair” refers to a pair ofhydrogen-bonded bases on opposite antiparallel strands of a nucleicacid. The rules of base pairing, which were first elaborated by Watsonand Crick, are well known to those of skill in the art. For example,these rules require that adenine (A) pairs with thymine (T) or uracil(U), and guanine (G) pairs with cytosine (C), with the complementarystrands anti-parallel to one another. As used herein, the term“Watson-Crick base pair” encompasses not only the standard AT, AU or GCbase pairs, but also base pairs formed between non-standard or modifiedbases of nucleotide analogs capable of hydrogen bonding to a standardbase or to another complementary non-standard base. One example of suchnon-standard Watson-Crick base pairing is the base pairing whichinvolves the nucleotide analog inosine, wherein its hypoxanthine baseforms two hydrogen bonds with adenine, cytosine or uracil of othernucleotides.

A “modified base” comprises one or more moieties that renders the basecleavable (a cleavable base) by one or more restriction enzymes. Theterms “modified base” and “modified deoxynucleotide triphiosphate” areat times used interchangeably. As will be appreciated by those of skillin the art a restriction enzyme can specifically recognize and cleave aparticular cleavable base (e.g., a single cleavable base), or canrecognize and cleave more than one cleavable base. A variety of modifiedbases are known in the art, such as modified purine bases (e.g.,Hypoxanthine, Xanthine, 7-Methylguanine, Inosine, Xanthosine,7-Methylguanosine) and modified pyrimidine bases (e.g.,5,6-Dihydrouracil, 5-Methylcytosine, 5-Hydroxymethylcytosine,Dihydrouridine, 5-Methylcytidine).

In some aspects, the modified base is a methylated, hydroxymethylated,and/or a fomylated base. In one aspect, the modified base is aformylated deoxynucleotide triphophate (dNTP). In other aspects, themodified base is a methylated dNTP. In some aspects, the modified baseis a methylated dNTP, a hydroxymethylated dNTP or a combination thereof.In some aspects, the one or more methylated deoxynucleotide triphophatesis one or more methylated cytosines, one or more hydroxymethylateddNTPs, one or more methylated adenosines or a combination thereof. Inother aspects, the one or more methylated cytosines is 5-methylcytosine, 5-hydroxymethyl cytosine, or a combination thereof. In yetother aspects, the one or more methylated adenosines is N6 methyladenosine.

In some aspects, the modified base is used in an amplification reaction.In some aspects, all or some of a (one or more) particular dNTP aremodified (e.g., methylated). In other aspects, about 1%, 2%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, etc. of a (one ormore) particular dNTP are modified. In other aspects, about 25% of a(one or more) particular dNTP are methylated.

As described herein the modified base is cleavable by one or morerestriction enzymes. As will be appreciated by those of skill in the arta restriction enzyme can specifically (selectively) recognize and cleavea particular cleavable base (e.g., a single cleavable base) to theexclusion of other cleavablebases, or can recognize and cleave more thanone cleavable base. In some aspects, the restriction enzyme digests anucleic acid sequence at the site of the modified base or at a site(loci) that is distant from the modified base (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, etc. basesaway from the modified based (e.g., methylated base)). In other aspects,the restriction enzyme can cleave a nucleotide sequence comprising amethylated base (e.g., a methyl specific restriction enzyme), anucleotide sequence comprising a hydroxymethylated base (e.g., ahydroxymethyl specific restriction enzyme), or a nucleotide sequencecomprising methylated bases and hydroxymethylated bases.

In some aspects, the restriction enzyme is capable of being deactivated(e.g., denatured). In some aspects, the restriction enzyme isdeactivated upon a change (e.g., increase; decrease) in temperature(e.g., heat labile; cold labile), a change (e.g., increase; decrease) inpH (e.g., pH labile), contact with a reagent (e.g., cofactors which candifferentially chelate (EGTA for Ca2+ and EDTA for Mg2+), or acombination thereof. In other aspects, the deactivation of therestriction enzyme is permanent. That is, in some aspects, once therestriction enzyme is deactivated, it cannot be reactivated (e.g.,renatured; brought back to its native (active) form). In aspects, inwhich more than one restriction enzyme is used, the first methylspecific restriction enzyme, the second methyl specific restrictionenzyme or both are deactivated upon a change in temperature, a change inpH, contact with a reagent (cofactors which can differentially chelated(EGTA for Ca2+ and EDTA for Mg2+).

As described herein, a sample comprising non target nucleic acid and/ortarget nucleic acid is contacted with a restriction enzyme that iscapable of being deactivated to produce a combination, and thecombination is maintained under conditions in which amplicons comprisingthe modified base which is recognized and cleavable by the restrictionenzyme are digested by the restriction enzyme prior to amplification. Asis known in the art, many amplification reactions comprise one or moresteps that involve an increase in temperature (e.g., to denature anucleic acid sequence such as double stranded DNA).

Thus, in some aspects, the restriction enzyme used in the methods of theinvention is deactivated upon a change in temperature. In a particularaspect, the restriction enzyme is deactivated upon an increase intemperature (e.g., a heat labile restriction enzyme), such as duringamplification of a nucleic acid sequence in an amplification reaction.Once the amplification reaction which includes a step that involves anincrease in temperature occurs, the restriction enzyme is deactivated.Thus, after amplification, amplicons which comprise the modified basewhich is recognized and cleavable by the restriction enzyme will not bedigested by the restriction enzyme since it is longer active.

In some aspects, the methyl specific restriction enzyme is MspJ1, FspE1,LpnPI, AspBHI, RlaI, SgrTI, AbaSI or a combination thereof.

As described herein, amplification or extension of a primer (e.g., DNAsynthesis) can be accomplished using a nucleic acid polymerase which iscapable of enzymatically-incorporating both standard (dNTPs) andmodified thiol deoxynucleotides (sdNTPs) into a growing nucleic acidstrand. As used herein, the phrase a “nucleic acid polymerase” or“nucleic acid polymerase enzyme” refers to an enzyme (e.g.,naturally-occurring, recombinant, synthetic) that catalyzes thetemplate-dependent polymerization of nucleoside triphosphates to formprimer extension products that are complementary to one of the nucleicacid strands of the template nucleic acid sequence. Numerous nucleicacid polymerases are known in the art and are commercially available. Insome aspects, the nucleic acid polymerases that are thermostable, i.e.,they retain function after being subjected to temperatures sufficient todenature annealed strands of complementary nucleic acids.

Suitable polymerases for the methods of the present invention includeany polymerase known in the art to be useful for recognizing andincorporating standard deoxynucleotides. Examples of such polymerasesare disclosed in Table 1 of U.S. Pat. No. 6,858,393, the contents ofwhich are incorporated herein by reference. Many polymerases are knownby those of skill in the art to possess a proof-reading, orexonucleolytic activity, which can result in digestion of 3′ ends thatare available for primer extension. In order to avoid this potentialproblem, it may be desirable to use polymerase enzyme which lack thisactivity (e.g., exonuclease-deficient polymerases, referred to herein asexo− polymerases). Such polymerases are well known to those of skill inthe art and include, for example, Klenow fragment of E. Coli DNApolymerase I, Sequenase, exo− Thermus aquaticus (Taq) DNA polymerase andexo− Bacillus stearothermophilus (Bst) DNA polymerase. In a particularembodiment, incorporation of deoxynucleotides, including modifieddeoxynucleotides (dNTPs), into a growing nucleic acid strand (e.g., DNA)is accomplished using a nucleic acid amplification reaction, such asPCR. Therefore, especially suitable polymerases for the methods of thepresent invention include those that are stable and function at hightemperatures (i.e., thermostable polymerases useful in PCR thermalcycling). Examples of such polymerases include, but are not limited to,Thermus aquaticus (Taq) DNA polymerase, TaqFS DNA polymerase,thermosequenase, Therminator DNA polymerase, Tth DNA polymerase, Pfu DNApolymerase, Q5 polymerase (New England Biolabs), and Vent (exo−)DNApolymerase. In another embodiment, incorporation of triphosphates intoRNA is accomplished using an RNA polymerase. Examples of RNA polymerasesinclude, but are not limited to, E. coli RNA polymerase, T7 RNApolymerase and T3 RNA polymerases.

The amplification reaction can further comprise one or more reagentsthat alters the nucleic acid's melting temperature. In some aspects, theone or more reagents comprises dimethyl sulfoxide (DMSO) Tri-methylglycine (Betaine) or a combination thereof.

As used herein, the phrase “target nucleic acid sequence” or “targetnucleotide sequence” can be any nucleotide sequence for which it isdesirable to obtain sequence information. As used herein, the term“nucleotide sequence” (target nucleotide sequence; non target nucleotidesequence) refers to a nucleic acid molecule (e.g., DNA, RNA) that isproduced by the incorporation of two or more nucleoside triphosphatesinto a single molecule via one or more covalent linkages (e.g., aphosphodiester bond, a phosphorothiolate linkage). A “target nucleotidesequence” can be any nucleotide sequence for which it is desirable toproduce or to obtain sequence information using the methods describedherein. The target nucleic acid sequence may be a polynucleotide oroligonucleotide sequence and may be single-stranded or double-stranded.Typically, when a target nucleic acid sequence is initially provided indouble-stranded form, the two strands subsequently will be separated(e.g., the DNA will be denatured). The target nucleic acid sequence alsomay be naturally-occurring, isolated or synthetic. Examples of suitabletarget nucleic acid sequence include, but are not limited to, genomicDNA, mitochondrial DNA, complementary DNA (cDNA), a PCR product andother amplified nucleotides. RNA may also be used as a target nucleicacid sequence. For example, RNA can be reverse transcribed to yieldcDNA, using methods known in the art such as RT-PCR. The target nucleicacid sequence may be used in any convenient form, according totechniques known in the art (e.g., isolated, cloned, amplified), and maybe prepared for the sequencing reaction, as desired, according totechniques known in the art. In a particular embodiment, the targetnucleic acid sequence comprises DNA. In a further embodiment, the targetnucleic acid sequence comprises a sense DNA strand and an antisense DNAstrand, wherein at least one primer is annealed to each strand. The nontarget nucleic acid, the target nucleic acid or both is single stranded,double stranded or a combination thereof. Examples of nucleic acidsequence include a nucleic acid library (e.g., RNA-Seq library, Chip-Seqlibrary, miRNA library, Hi-C library), genomic nucleic acid,mitochondrial nucleic acid or a combination thereof.

A nucleotide sequence can be obtained from any of a variety of sources.For example, DNA or RNA may be isolated from a sample, which may beobtained or derived from a subject.

The word “sample” is used in a broad sense to denote any source of anucleotide sequence on which sequence determination is to be performed.The source of a sample may be of any viral, prokaryotic,archaebacterial, or eukaryotic species. The sample may be blood oranother bodily fluid containing cells; sperm; and a biopsy (e.g.,tissue) sample, among others.

As used herein, the term “primer” refers to an oligonucleotide, which iscapable of acting as a point for the initiation of synthesis of a primerextension product that is complementary to the template polynucleotidesequence. The primer may occur naturally, as in a purified restrictiondigest, or be produced synthetically. The appropriate length of a primerdepends on the intended use of the primer, but typically ranges fromabout 5 to about 100; from about 5 to about 75; from about 5 to about50; from about 10 to about 35; from about 18 to about 22 nucleotides. Aprimer need not reflect the exact sequence of the template but must besufficiently complementary to hybridize with a template for primerelongation to occur, i.e., the primer is sufficiently complementary tothe template polynucleotide sequence such that the primer will anneal tothe template under conditions that permit primer extension. As usedherein, the phrase “conditions in which that target nucleic acidsequence is amplified” or “conditions that permit primer extension”refers to those conditions, e.g., salt concentration (metallic andnon-metallic salts), pH, temperature, and necessary cofactorconcentration, among others, under which a given polymerase enzymecatalyzes the extension of an annealed primer. Conditions for the primerextension activity of a wide range of polymerase enzymes are known inthe art. As one example, conditions permitting the extension of anucleic acid primer by Taq polymerase include the following (for anygiven enzyme, there can and often will be more than one set of suchconditions): reactions are conducted in a buffer containing 50 mM KCl,10 mM Tris (pH 8.3), 4 mM MgCl2, (200 mM of one or more dNTPs and/or achain terminator may be included, depending upon the type of primerextension or sequencing being performed); reactions are performed at 72°C.

It will be clear to persons skilled in the art that the size of theprimer and the stability of hybridization will be dependent to somedegree on the ratio of A-T to C-G base pairings, since more hydrogenbonding is available in a C-G pairing. Also, the skilled person willconsider the degree of homology between the extension primer to otherparts of the amplified sequence and choose the degree of stringencyaccordingly. Guidance for such routine experimentation can be found inthe literature, for example, Molecular Cloning: a laboratory manual bySambrook, J., Fritsch E. F. and Maniatis, T. (1989).

Conditions for amplification will vary depending upon the type ofsequence being amplified and the type of amplification being used.Examples of conditions under which an amplification reaction ismaintained in order to amplify a nucleic acid sequence include one ormore amplification cycles which comprises 98° C. for 20 seconds, 60° C.for 15 seconds, 72° C. for 60 seconds; 12° C. for 60 seconds, 98° C. for20 seconds, 60° C. for 15 seconds, 72° C. for 60 seconds, 12 sequencingcycles at 98° C. for 20 seconds, 72° C. for 3 minutes; an initial 1minute denaturization at 94° C. followed by 30 cycles of 98° C. at 10 s,68° C. for 15 minutes; performance of a final 72° C. 10 minute extensionprior to 4° C. hold; 12 Cycles of 72° C. for 3 minutes, 98° C. for 30seconds, 12 cycles of 98° C. for 10 seconds, 63° C. for 30 seconds, 72°C. for 1 minute. In some aspects, the amplification reaction cancomprise a heat kill which is followed by a Phi-29 isothermalincorporation (e.g., 80° C./20 minutes to heat kill the MspJI/AbaSI andthen add Phi29 for methylated isothermal amp at 37° C., Bst polymeraseisothermal amps

In some aspects, deoxyinosine triphosphate (dITP) is used in conjunctionwith Endonuclease VIII which specifically cleaves Inosine.

In addition to decontamination, the hemi-stranding aspects can be usedto sequence specific strands of a library.

Methods that use digestible nucleotides have been described (Hartley andRashtchian 1993). If one desires to replicate a library of singlestranded DNA (e.g., an oligo pool) but needed to remove any required PCRprimer sites required for amplification, deoxyuridine triphosphate(dUTP) and dITP nucleotides are poor choices as they will beincorporated into the amplicon randomly and not be constrained to theprimer sequences. Uracil or inosine could also be sequenced into theprimer sequences but these cleavage signals would not be replicated inPCR on subsequent PCR cycles as polymerases incorporate nativenucleotides over these bases in PCR. Additionally, these cleavagesignals direct enzymes that only cleave one strand of DNA leaving anoverhang that needs subsequent and careful end repair. The use of doublestranded restriction enzymes has been described but due to the larger(4-20 base pair) recognition signals in restriction enzymes, its notalways possible to have a restriction enzyme manage the cleavage of allamplification primers. In addition there is always the concern of therestriction enzyme digesting the target sequence to be amplified. Asignal would preferably have a small recognition signal (1-2 bases),cleave both strands preferably remotely, have affinity for variouslaboratory capture reagents, and be specific for the primer sequencesand non-existent in any target sequence.

As described herein, modified bases (e.g., methyl dCTP) in conjunctionwith restriction enzymes that cleave the modified bases (e.g., MspJI)uniquely meets these requirements and differs from other amplificationtechniques. In one aspect, target sequences are amplified withtransliterated sequence identity which provides for easy decontaminationtechniques.

An aspect of the methods provided herein is exemplified usingmethyl-dCTP as a replacement for dCTP. Previously, Wong et al describedPCR with 5-methyl-dCTP to screen for “methyl sensitive restrictionendonucleases” which were used to screen for restriction endonucleasesactivities which were blocked by the presence of a methylated cytosine(Wong and McClelland 1991). However, at the time of Wong, “methylspecific restriction enzymes”, also known as “methyl dependentrestriction enzymes”, had not been discovered (Cohen-Karni et al.;Horton et al.; Zheng et al.).

Described herein is the use of the methyl dependent enzymes (e.g.,MspJI, AbaSI enzyme) in combination with one or more methylated dNTPS(e.g., 5-methyl-dCTP; 5-hydroxymethyl dCTP) for amplification methodssuch as PCR. The methods described herein can be used as a replacementfor UNG. One benefit of a methylated dNTP embodiment over the use ofuracil is that more polymerases are literate with methylated dNTP.Additionally, enzymes like Dnmt1 exist which can replicate the methylgroup onto the opposite strand if optionally required. Additionally,enzymes such as MspJI are a single enzyme system which can digest DNA onboth strands with a single methylated cytosine signal and will notdigest DNA with unmethylated cytosine.

In contrast, UNG only removes the Uracil nucleobase by digesting theglycosic bond and thus requires other enzymes such as Endo8 to excisethe ribose, and polynucleotide kinase to remove phosphates. After using3 enzymes one is still only left with a single stranded digestion andone must remove the other strand with T7 exonuclease.

In another aspect, the invention is directed to a method, referred toherein as “Ephemeral Primer Amplification” or EPA, in which methyl dCTPand MspJI are used to replicate oligo libraries.

Oligonucleotides are staples in the DNA diagnostic and DNA sequencingfields. Exome sequencing requires synthesizing 100 s to 100,000 soligonucleotides to use as baits for capturing targeted regions of DNAfor sequencing. DNA synthesis costs are still expensive often costingseveral dollars per oligonucleotide. For this reason there exists a needto immortalize or amplify an oligonucleotide (or Probe) library.

Traditional approaches to amplification utilize PCR or Rolling CircleAmplification (RCA). All amplification techniques require PCR primersequences. These additional PCR primer sites are unwanted DNA sequenceson the oligonucleotides probes.

Also described herein is a method to attach universal PCR primer sitesto ssDNA oligos and to subsequently remove them after amplification torestore the Oligo nucleotide library to its native form afteramplification is described.

Fire et al describe the use of 5′ independent ligation of RNA. (Pak andFire 2007) This method relies on the use of T4Rnl ligase which is atemplate independent ligase. This ligase requires RNA as the 3′ acceptormolecule but can utilize DNA as the 5′ phosphate donor molecule.Zhelkovsky describe a ligase that can complete step 3 of ligation whilebeing dysfunctional for step 1 and 2. This enables the ligation of 5′Pre-adenylated oligonucleotides. (Zhelkovsky and McReynolds). As aresult this ligase is very efficient at ligation and does not requireATP. ATP can be a competitive inhibitor to ligation as too much ATP candrive the ligation reaction backwards leaving many adenylated oligos asa side product. Zhelkovsky also describes a novel RNA ligase fromMethanobacterium thermoautotrophicum (Zhelkovsky and McReynolds). Thisligase can ligate single stranded DNA as both an acceptor molecule and adonor molecule in a template independent manner. Kool describes atemplate independent method for ligation but it requires modifiedoligonucleotides to perform chemical ligation and not all targetoligonucleotides have this desired functional group (Xu and Kool 1997).

With Zhelkovskys' novel ligases one can now imagine ligating primers onthe 3′ end of a DNA probe library. Li also describe a ligase which cando this given a 10,000 fold excess of donor over acceptor molecules (Liand Weeks 2006). With the proper donor primer design (utilizing ablocked 3′ end of the donor primer), double stranding this probe libraryresults in only 1 active 3′ hydroxyl on the newly generated secondstrand. This hydroxyl can become the target for the second primer siteto be added. This approach is very analogous to Fire's technique formaking 5′ independent cloning of RNA except it can now be performed onDNA. The inventive aspect of this method is the combination of thistechnique with an amplification strategy that removes its PCR primersafter amplification and single strands the amplified library to resultin a identical but amplified oligonucleotide probe library.

Once Primer sites have been added to both ends of a probe library, PCRcan be performed. There are several ways to remove primer sites afterPCR but they all have current undesirable properties. Putting Uracils inthe primers is one method of digesting the primers after PCR with Uracilspecific nucleases. This suffers from being a multi-enzyme digestion asUDG only digests the glycosic bond on one strand. Restriction enzymesare often used to cut the primers off but these suffer from alsopotentially cutting the internal oligo one is attempting to replicate.Methyl sensitive restriction enzymes can be deployed but they often cutboth methylated and non-methylated DNA. Recently a Methyl Dependentclass of restriction enzymes have been described (Zheng et al.).Positioning these methyl dependent signals in the PCR primer enables amethod which can remove the primer sites after amplification with asingle step while avoiding internal digests and multiple enzyme endrepair step with the other two methods. See FIGS. 1-4.

Articles such as “a”, “an”, “the” and the like, may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext.

The phrase “and/or” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined. Multiple elements listed with “and/or” should be construed inthe same fashion, i.e., “one or more” of the elements so conjoined.Other elements may optionally be present other than the elementsspecifically identified by the “and/or” clause. As used herein in thespecification and in the claims, “or” should be understood to have thesame meaning as “and/or” as defined above. For example, when used in alist of elements, “or” or “and/or” shall be interpreted as beinginclusive, i.e., the inclusion of at least one, but optionally more thanone, of list of elements, and, optionally, additional unlisted elements.Only terms clearly indicative to the contrary, such as “only one of” or“exactly one of” will refer to the inclusion of exactly one element of anumber or list of elements. Thus claims that include “or” between one ormore members of a group are considered satisfied if one, more than one,or all of the group members are present, employed in, or otherwiserelevant to a given product or process unless indicated to the contrary.Embodiments are provided in which exactly one member of the group ispresent, employed in, or otherwise relevant to a given product orprocess. Embodiments are provided in which more than one, or all of thegroup members are present, employed in, or otherwise relevant to a givenproduct or process. Any one or more claims may be amended to explicitlyexclude any embodiment, aspect, feature, element, or characteristic, orany combination thereof.

EXEMPLIFICATION Example 1 EPA

Materials and Method for EPA—

Oligos ordered (SEQ ID NO: 3)Oligo 1-/5PHOS/ATC GAC AAC AAC TCT CCG TCC TCC GTG CG/3SpC3/-ORDERED (SEQ ID NO: 4) Oligo 2-CGC ACG GAG GA/iMe-dC/GGA GAG TTG TTGTCG AT-ORDERED  (SEQ ID NO: 5)Oligo 3-TTC ACT CCT AGC TT/iMe-dC/TCA TGT AGA GAC TCA C/iBiodT/T GCC (SEQ ID NO: 6) Oligo 4-/5Phos/GG CAA GTG AGT CTC TAC ATG AGAAGC TAG GAG TGA A/3 SpC3/  ILMN Methyl Primer 1.0 (SEQ ID NO: 7)AATGATACGGCGACCACCGAGATCTACACTCTTTC/iMe-dC/ CTACACGA-ORDERED ILMN Methyl Primer 2.0 (SEQ ID NO: 8)CAAGCAGAAGACGG/iMe-dC/ATACGAGAT-ORDERED 

Adenylation of Phosphorylated Oligos.

This can be performed by IDT or Enzymatically with reagents from NEB.

1 ul (100 um Oligo 1)

2 ul 1 mM ATP

2 ul 10× Adenylation buffer

2 ul Mth Ligase

13 ul ddH20

1 hour 65° C.

5 min 85° C. heat kill.

Methods for decontamination procedures.

A library for Illumina sequencing was made utilizing the Nextera Kitaccording to the manufacturers instruction. This library was then PCRamplified with native nucleotides and compared to amplification wheredTTP was replaced with dUTP. Kapa Uracil+polymerase was utilized. SeeFIGS. 5A-5C.

0.5 ul 10 uM Primer1.0

0.5 ul 10 uM Primer2.0

2 ul 2 mM dNTP (dUTP was swapped out at the same concentration as dTTP)

5 ul 5× Kapa Uracil+buffer

3 ul DNA (Post Ampured Nextera amplified DNA eluted in 20 ul)

8 ul ddH20

1 ul Kapa Uracil+Polymerase (1 U/ul)

20 ul Total Reaction

PCR was performed using the following thermal cycling conditions.

1) 95° C. for 2 mins

2) 98° C. for 20 sec

3) 60° C. for 15 sec

4) 72° C. for 1 min

5) Go to 2 for 12 cycles

6) 72° C. for 3 mins

These PCR products were then purified with Ampure according to themanufacturer's recommendations and eluted in 25 ul of ddH20.

1 ul of the eluent was then run on an Agilent Bioanalyzer HS chip tosupply the above electropherograms.

Although PCR amplification is more efficient with native nucleotides,complete replacement with Uracil can be amplified with Uracil tolerantpolymerases.

5 ul of the Post Ampure Purified Libraries were digested to confirmamplification with dUTP.

5 ul DNA

1 ul UDG

1 ul FpG

1 ul LifeTech FuPa reagent

2 ul 10×UDG buffer

10 ul H20

37° C. for 30 minutes

Ampure with 30 ul Ampure

Elute in 30 ul ddH20

Load 1 ul on Agilent Bioanalyzer HS chip.

Example 2 Use of 5′Methyl dCTP in PCR and MspJI Digestion forDecontamination

PhiX Library was amplified with and without 5methyl dCTP spiked in.

17 ul of Q5 Polymerase (NEB)

2 ul of 10 uM ILMN 1.0 and ILMN 2.0 Primers

2 ul of 1:100 PhiX control library

2 ul 5 mM 5methyl dCTP

11 ul ddH20

34 ul Reaction

12 cycles of 12° C. 60

2) 98° C. for 20 sec

3) 60° C. for 15 sec

4) 72° C. for 1 min

5) Go to 2 for 12 cycles

6) 72° C. for 3 mins

17 ul of the reaction was Ampured with 30 ul. Eluted in 20 ul and 1 ulloaded on an Agilent HS chip. A noticeable gel shift is seen with themethylated amplification. See FIG. 6.

These libraries were both digested with MspJI

10 ul of Amplification product

3 ul 10×NEB buffer 4

1 ul Enzyme Activator

1 ul 100×BSA

14 ul ddH20

1 ul MspJI

TipMix and incubate for 37° C. for 1.5 hours.

Ampure 15 ul of reaction with 30 ul of Ampure. Elute in 15 ul. Load 1 ulon Agilent Bioanalyzer.

The electropherograms in FIG. 7 demonstrate that PCR can be performedwith 5′ methyl dCTP and that these PCR products can be specificallytargeted with methyl specific nucleases like MspJI.

Will methylated libraries amplify and sequence on the Illumina MiSeqsequencer?

Two PhiX control libraries were amplified as described above. The onlymodification was the inclusion of a pool of 6 different DNA barcodes forthe Control conditions (barcodes 1-6) and 6 different DNA barcodes(7-12) for the 5methyl dCTP amplified library. These libraries werepurified with Ampure and loaded onto the MiSeq according to themanufacturers instructions. 50 bp reads were generated and 6 bases weresequenced for the barcode. Reads were demultiplexed and counted. 4.35million reads were observed with the control conditions and 4.26M readswere observed with the 5methyl dCTP libraries suggesting the IlluminaMiseq can sequence methylated Cytosines in the templates. See FIG. 8.

Can Agilents Haloplex capture system utilize 5methyl dCTP EPA andproduce sequence?

Eluted Haloplex NaOH in 40 ul instead of the recommended 25 ul. Took 20ul and amplified it with the recommended conditions using Herculase PCR.Used remaining 20 ul for 5methyl dCTP PCR with Q5 polymerase.

1) 20 ul DNA

2) 1 ul Primer 1.0 (25 uM)

3) 1 ul Primer 2.0 (25 uM)

4) 0.5 ul 2M Acetic Acid (neutralize NaOH)

5) 2 ul 5 mM 5methyl dCTP

6) 25 ul Q5 2×PCR premix (NEB)

7) Cycle using 18 cycle conditions used for control

98° C. 2:00

98° C. 30 sec

60° C. 30 sec

72° C. 1:00 min

Go to step 2 17 more times

72° C. 4 mins

10° C. forever

Ampure using 1.2× Ampure (60 ul onto 50 ul reaction)

Elute DNA in 40 ul ddH20.

Load 1 ul onto the Agilent HS Bioanalyzer

1 ul of a 1:10 dilution of a 50 ul New England Biolabs Q5 polymeraseamplifying with 0.2 mM 5methyl dCTP supplement. Target library wascaptured with a modified Agilent Haloplex reagent. Library contains 327genes from Courtagens EpiSEEK panel. This clinical test sequences over5,000 exons to 200× coverage or more. See FIG. 9.

A control library from the same patient was sequenced using the standardprotocol utilizing native dCTP. Electropherograms look similar.Methylated library delivered 4.82 ng/ul while the control librarydelivered 8.0 ng.ul. Libraries were sequenced on the Illumina MiSeq tounderstand coverage bias. See FIG. 10.

Libraries were barcoded and loaded on to a MiSeq generating 1.327Mclusters per mm^2. No sign of inefficient clustering is seen in the CChannel. 10 Gb run is expected. See FIG. 11.

5 Million 250 bp reads were generated from 2 patients (490 and 820)using an ILMN MiSeq sequencer with V2 chemistry. Patient 820 wassequenced with both dCTP (purple) and 5-methyl-dCTP (red). Resultsdemonstrate that over 95% of the 5,000 exons targets are sequenced to20× coverage or higher. Courtagens Clinical cutoff for acceptable datais 90% of the targets covered at least 10× or higher in coverage. SeeFIG. 12.

Example 3 Déjà vu PCR: DREAMing and Re-DREAMing PCR Methods

Described herein is a PCR method that utilizes six nucleotides in PCRwith two methyl sensitive restriction enzymes that respectively digestthese additional nucleotides. Use of this enzyme and nucleotidecombination enabled what is termed herein a “DNA diode” where DNA canadvance in a laboratory in only one direction and cannot feedback intoupstream assays. Aspects of this method that enable consecutiveamplification with the introduction of a 5th and 6th base whilesimultaneously providing mitochondrial DNA enrichment are described.

Methods

Long-Range PCR

PCR setup utilized forward and reverse primers for the 16 kb product:mtPCR6F-321-5′ TGGCCACAGCACTTAAACACATCTC 3′ (SEQ ID NO: 9) andmtPCR6R-16191-5′ TGCTGTACTTGCTTGTAAGCATGGG3′ (SEQ ID NO: 10). PCR wasperformed utilizing 15 ng of gDNA (10 ng/ul). Reaction setup included1.5 ul of DNA, 5.0 ul of 10× LA PCR Buffer II, 0.5 ul TaKaRa LA Taq DNApolymerase, 10.65 ul ddH20, and 0.125 ul (50 uM) of each primer with 8.0ul dNTP mixture (2.5 mM each dNTP where a ratio of 75:25 dCTP:5me-dCTP).The 50 ul PCR reaction was cycled with an initial 1 minutedenaturization at 94° C. and is followed by 30 cycles of 98° C. at 10 s,68° C. for 15 minutes. A final 72° C. 10 minute extension is performedprior to 4° C. hold. PCR products are purified using 75 ul of Ampure(Beckman Genomics).

Nextera Reaction and 5-hydroxymethylcytosine PCR

3 ul (2.5 ng/ul) of the purified LR-PCR product is used in a 10 ulNextera reaction ( 1/20th×) utilizing 5.0 ul TD, 0.25 ul of TDE, 1.75 ulddH20 (acronyms according to manufacturers instructions). Samples areincubated for 30 minutes at 55° C. followed by a 15 ul Ampurepurification. Products are eluted in 25 ul of dH20 and 10 ul of eluentare used for Nextera PCR with 0.75 ul of each 10 uM primer, 1.25 ul ofeach Illumina index, 20 ul of 2×Q5 polymerase (New England Biolabs) and0.75 ul of 5 mM 5-hydroxymethylcytosine (Trilink) with a 4% final DMSO.12 Cycles of PCR are performed with the following cycling protocol: 72°C. for 3 minutes, 98° C. for 30 seconds, 12 cycles of 98° C. for 10seconds, 63° C. for 30 seconds, 72° C. for 1 minute. PCR products arepurified using 52.5 ul of Ampure. These products are optionally sizeselected with a SAGE Sciences Pippin PrepII system in the 600-800 bpsize range for 2×250 bp sequencing on a MiSeq V2 sequencer from Illuminaaccording to the manufacturers instructions.

Decontamination

MspJI digestion is performed with 10 ul DNA (6-8 ng/ul), 1.5 ul 10×buffer, 1.0 ul Activator, 1.5 ul 10×BSA, 0.5 ul MspJI at 37 C for 30minutes. The sample is heat killed at 65° C. for 20 minutes beforeinitiating PCR.

AbaSI digestion is performed with 10 ul DNA (6-8 ng/ul), 1.5 ul 10×buffer, 1.0 ul AbaSI, 2.5 ul ddH20 at 25° C. for 1 hour. The sample isheat killed at 65° C. for 20 minutes before initiating PCR. FIG. 16demonstrates the decontamination with AbaSI.

Enrichment Ascertainment

Haloplex assays were designed and amplified according to themanufacturers version 2 instructions (Agilent). MspJI digestion wasperformed as described above but with 1 unit of enzyme. Experiments wereDNA barcoded and sequenced with Illumina Miseq V2 sequencer with 2×250bp reads to ensure high mapping quality. All reads were mapped withBowtie2 and coverage calculations were performed with BEDTools aspreviously described (McKernan in press).

The control samples demonstrated a M:N ratio of 12.3. Mitochondrial DNAis known to be in several hundred to thousand copies per cell and theM:N amplicon target ratio is 16 kb/246 kb. Since the 246 kb nucleartarget is only n=2 in copy number next to an estimated n=500 forMitochondria, we can adjust the formula to M(n-mito)/N(n-nuc) to get 16kb*500/246 kb*2 with an expect read ratio of 16. The M:N ratio of the 3units of MspJI treated gDNA samples is over twice as high (27.3) as thecontrols (FIG. 18). To further confirm these results we used magneticparticles (New England Biolabs, EpiMark) with Methyl Binding Domain(MBD) to methyl capture and sequence a given sample to demonstrate farlower M:N ratios. The MBD particles deliver confirmatory evidence fordifferential methylation between Mitochondrial and Nuclear DNA (FIG.18).

Results

Consecutive amplification utilizes a 6th base.

Several clinically relevant next generation sequencing assays require atleast two serial amplification steps. Techniques designed to identifylong range genomic phasing often employ whole genome amplification (WGA)before using a more directed PCR approach. In addition, some exomecapture techniques require a pre-capture PCR and a post-capture PCRstep. Provided herein is a serial PCR which includes an amplificationstep that comprises a decontaminating methylated cytosine. Specifically,the method is demonstrated herein using 16 kb long range PCR (LR-PCR) toamplify the whole mitochondrial genome for subsequenttransposon-mediated library construction, followed by a 12-cycleamplification step (Nextera PCR reaction) using universal Illuminaprimers.

The serial amplification procedures provided herein utilize universalprimers and two different digestible nucleotides, e.g., 5me-dCTP and5hme-dCTP (Trilink), for exclusive use in respective amplifications. Theenzyme AbaSI (NEB) selectively digests 5-hydroxymethylcytosine withoutdigesting 5-methylcytosine.

Decontamination techniques work best when the target to be amplified isdifferent than the product or potential contaminant. If 5me-dCTP existsin the first LR-PCR product, one cannot use MspJI to decontaminate thesecond Nextera PCR reaction, as MspJI is a methyl-specific restrictionenzyme and will digest both the substrate 16 kb target amplicon and anypotentially contaminating Nextera PCR products. As shown herein, inorder for decontamination to be effective the post-amplified (e.g.,Nextera) contaminants require a nucleotide (5-hydroxymethylcytosine)that does not exist in the 5-methylcytosine LR-PCR DNA (FIG. 13).

The described LR-PCR has site-specific primers, thus, contaminants froma Nextera PCR reaction with different universal primers are less likelyto create amplifiable contamination. Nevertheless, these Nexteralibraries contain mitochondrial DNA, a small portion of which iscomplementary to the LR-PCR primers and secondary amplificationartifacts can in fact amplify and impair heteroplasmy detection. Inaddition to this source of background, deleted mitochondria from otherclinical samples can hyper-amplify if co-present with clinical mtDNAwhich is significantly longer in length. FIG. 14 demonstrates how apatient with a 4.5 kb mitochondrial deletion known to be associated withKearns-Sayre syndrome can hyper-amplify (10×) in a foreground of 16.6 Kbtarget amplification. Thus, the two sources of potential contaminationunderscore the need for decontamination techniques.

Long Range PCR Considerations

The use of LR-PCR for massively parallel mitochondrial sequencing hasproven to have the most sensitive heteroplasmy and large deletiondetection. This is largely due to LR-PCR's ability to deliver uniformcoverage and to limit the amplification of similar Nuclear MiTochondrialor NUMTs sequences found with methods that use hybridization capturetechniques. Nevertheless, LR-PCR methods can be hindered by jumping PCRartifacts with NUMTs and often the heteroplasmy sensitivity is limitedto 1% allele frequencies, despite the fact that sequencing techniquescan deliver accurate allele frequencies far below this. Since 90% ofmtDNA deletions are larger than 2 kb, LR-PCR methods are also prone tohyper-amplification of clinically relevant deleted mtDNA samples. Thishyper-amplification is an advantage for clinical sensitivity but alsopresents a leveraged contamination risk if background deletedmitochondrial samples contaminate other clinical samples.

To address this, described herein is a decontamination approach thatconcurrently depletes NUMTs from the sample. Prior to initiation of PCR,the sample is digested with MspJI which digests hyper-methylated dsDNAthat can otherwise contaminate the LR-PCR. Exhaustive bisulfitesequencing of mitochondria in several tissues has demonstrated completelack mitochondrial DNA methylation, while NUMTs are rapidly methylatedin the nuclear genome. This suggests methyl specific restrictiondigestion can selectively digest NUMTs and render them non-amplifiable.

During the first LR-PCR amplification a mixture of dCTP and 5-methyldCTP was used. During the second Nextera PCR a mixture of dCTP and5-hydroxymethylcytosine was used. Since MspJI digests both5-methylcytosine and 5-hydroxymethylcytosine, it decontaminated theLR-PCR reaction setup of both past LR-PCR product and past Nextera PCRproduct contaminants while also digesting NUMTs gDNA. MspJI has apreference of double-stranded DNA over single-stranded DNA and thispreference may alter a given application.

After the first LR-PCR and prior to the second Nextera PCR AbaSI wasused to digest contaminants as this enzyme only digests5-hydroxymethylcytosine, leaving 5-methylcytosine or cytosine intact. Inthis case, AbaSI only digested PCR products that contaminated thepre-Nextera sample from the post secondary PCR process (FIG. 15). Thesecond PCR usually contains universal sequencing primers producing smallproducts (700 bp) desired by the limitations of current sequencers.These smaller PCR products can hyper-amplify due to cold PCR or otherselective amplification biases and as a result can be over represented.Hyper-amplification of contaminants in PCR are the largest risk in aclinical laboratory testing for heteroplasmy.

Decontamination and Optimal Sequencing Performance

Since 5-methylcytosine alters the Tm of DNA by 0.5° C. per methylatedcytosine, optimizations to the PCR conditions were explored. Previousstudies with DREAM PCR demonstrated decaying sequencing coverage withincreasing concentrations of 5-methyl dCTP. (McKernan et al) Raising theannealing and denaturization temperatures to compensate for5-methyl-dCTP's impact on Tm exposes DNA to hydrolytic damage. As aresult, methods that alter the solvation and melting temperature withoutintroducing thermal damage to the DNA were pursued. It was found thatabout a 4% final concentration of DMSO provided optimal sequencingcoverage (FIG. 17) equal to non-methylated amplification controls.

Decontamination was measured by spiking in known amounts of DNAcontaminant from a different mitochondrial haplogroup. These sampleswere treated with the respective enzymes and deeply sequenced (10,000×)to measure the percent heteroplasmy of the sample at the haplogroupspecific loci. A simple 1 hr digestion was able to remove a 50 foldexcess of contaminating DNA (FIG. 19). This assay is limited in that itis only measuring contamination at <40 haplogroup specific loci.

Mitochondrial Enrichment

To measure the mitochondrial DNA enrichment a Haloplex assay thattargeted both the entire mitochondrial genome (320 amplicons) andseveral nuclear genes in parallel (13,060 amplicons) was designed.Genomic DNA was purified and treated with and with out MspJI digestion(0, 0.3, 0.5, 1, 2, 3 units of MspJI enzyme). These libraries were thensequences, and the reads were mapped to hg19 including the mitochondrialgenome to measure the ratio of reads mapping to nuclear versusmitochondrial targets. This mapped read ratio was termed the M:N ratioand was used to estimate enrichment. The M:N ratio in the control samplewas 12.3 while the MspJI digested sample had a M:N ratio of 27.3,demonstrating an enrichment of mitochondrial DNA through the digestionof methylated gDNA. Quantitative PCR was performed to confirm the M:Nratio of the source DNA (FIG. 18).

Discussion

These results demonstrate additional utility of DREAM PCR indecontaminating more complex amplification procedures than describedpreviously (REF). In addition the importance of such decontaminationtechniques for mitochondrial sequencing and the impact suppressing largedeletion hyper-amplification is underscored. Also demonstrated herein isa beneficial enrichment of mtDNA by leveraging the lack of methylationin mitochondrial DNA. This addresses a problem with NUMTs contaminatingmany next-generation mitochondrial sequencing assays previouslydescribed and likely opens the field for accurate sub percentageheteroplasmy sensitivity.

These results likely have relevance for accurate sequencing in anysample that demands low allele frequency quantification likeheterogeneous biopsies. Likewise, the results underscore the value ingenerating ephemeral PCR products. With recent concerns over DNAconfidentiality and the ease of de-identification of DNA samples, dataencryption is becoming a standard in clinical laboratory data managementto prevent in-silico contamination or disclosure of DNA sequence.Considering physical DNA can be harvested from 50,000 year old samples,a clinical laboratory's trash is a confidentiality exposure point if DNAis not digested or destroyed during testing. Thus methods that eliminateDNA from a clinical laboratory offer attractive and responsiblefeatures. In summary, a method that improves DREAM PCR sequencingperformance while providing more freedom to operate concurrently with amore responsible clinical management of patient DNA is provided herein.

The teachings of all patents, published applications and referencescited herein are incorporated by reference in their entirety.

While this invention has been particularly shown and described withreferences to example embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method of removing amplicons of a non target nucleic acid having one or more methylated cytosines from a sample wherein the sample comprises amplicons of the non target nucleic acid and genomic DNA that includes a target nucleic acid to be amplified, the target nucleic acid having one or more methylated cytosines, comprising: a) contacting the sample with a composition comprising a methyl specific restriction enzyme that specifically cleaves a nucleic acid having one or more methylated cytosines, wherein the enzyme does not cleave an unmethylated cytosine and is capable of being deactivated, thereby producing a combination; b) maintaining the combination under conditions in which the amplicons of the non target nucleic acid are digested by the methyl specific restriction enzyme prior to amplification of the genomic DNA that includes the target nucleic acid; and c) amplifying the genomic DNA that includes the target nucleic acid thereby producing amplicons of the target nucleic acid, wherein the amplicons of the non target nucleic acid are removed from the sample comprising the genomic DNA that includes the target nucleic acid to be amplified.
 2. The method of claim 1, wherein the methyl specific restriction enzyme is deactivated upon a change in temperature, a change in pH, contact with a reagent or a combination thereof.
 3. The method of claim 1, wherein the composition of a) further comprises (i) deoxynucleotide triphosphates (dNTPs) comprising dATP, dTTP, dGTP, and dCTP (ii) a nucleic acid polymerase, and (iii) one or more primers that is complementary to a portion of the target nucleic acid.
 4. The method of claim 3, wherein one or more of the deoxynucleotide triphosphates are methylated cytosines.
 5. The method of claim 4, wherein the one or more methylated cytosines is 5-methyl cytosine, 5-hydroxymethyl cytosine, or a combination thereof.
 6. The method of claim 4, wherein the amplicons of the target nucleic acid are further contacted with a methyl specific restriction enzyme, thereby producing a combination; and maintaining the combination under conditions in which the amplicons of the target nucleic acid are digested by the methyl specific restriction enzyme.
 7. The method of claim 6, wherein the methyl specific restriction enzyme that is contacted with the amplicons of the target nucleic acid (i) is identical to the methyl specific restriction enzyme that is contacted with the amplicons of the non target nucleic acid or (ii) is a different methyl specific restriction enzyme than the methyl specific restriction enzyme that is contacted with the amplicons of the non target nucleic acid.
 8. The method of claim 6, wherein the amplicons of the target nucleic acid are further contacted with a methyl specific restriction enzyme prior to amplification of a second target nucleic acid.
 9. The method of claim 1, wherein the methyl specific restriction enzyme is MspJ1, FspE1, LpnPI, AspBHI, RlaI, SgrTI, AbaSI or a combination thereof.
 10. The method of claim 1, wherein the composition of a) further comprises one or more reagents that alters the melting temperature of the non target nucleic acid or the target nucleic acid or combinations thereof.
 11. The method of claim 10, wherein the one or more reagents comprises dimethyl sulfoxide (DMSO) Tri-methyl glycine (Betaine) or a combination thereof.
 12. The method of claim 1, wherein the non target nucleic acid is single stranded, double stranded or a combination thereof.
 13. The method of claim 1, wherein the non-target nucleic acid is a nucleic acid library, genomic nucleic acid, mitochondrial nucleic acid or a combination thereof.
 14. The method of claim 12, wherein the double stranded nucleic acid is denatured prior to amplification.
 15. The method of claim 1, wherein the target nucleic acid is amplified for at least 1 amplification cycles wherein each amplification cycle comprises 98° C. for 20 seconds, 60° C. for 15 seconds, 72° C. for 60 seconds, 12 cycles at 98° C. for 20 seconds, and 72° C. for 3 minutes. 